Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized

L'appui technique aux artisans

It has changed overtime with new legislation (such as VAT and compulsory management training sessions), with changes in economic environment and as the result of bargaining with local units of government. However even if there is more advice on economic and marketing decisions, most of the support is in technological training

L'appui technique aux artisans

It has changed overtime with new legislation (such as VAT and compulsory management training sessions), with changes in economic environment and as the result of bargaining with local units of government. However even if there is more advice on economic and marketing decisions, most of the support is in technological training

The effects of stimulus complexity on the preattentive processing of self-generated and nonself voices: an ERP study

The ability to differentiate one's own voice from the voice of somebody else plays a critical role in successful verbal self-monitoring processes and in communication. However, most of the existing studies have only focused on the sensory correlates of self-generated voice processing, whereas the effects of attentional demands and stimulus complexity on self-generated voice processing remain largely unknown. In this study, we investigated the effects of stimulus complexity on the preattentive processing of self and nonself voice stimuli. Event-related potentials (ERPs) were recorded from 17 healthy males who watched a silent movie while ignoring prerecorded self-generated (SGV) and nonself (NSV) voice stimuli, consisting of a vocalization (vocalization category condition: VCC) or of a disyllabic word (word category condition: WCC). All voice stimuli were presented as standard and deviant events in four distinct oddball sequences. The mismatch negativity (MMN) ERP component peaked earlier for NSV than for SGV stimuli. Moreover, when compared with SGV stimuli, the P3a amplitude was increased for NSV stimuli in the VCC only, whereas in the WCC no significant differences were found between the two voice types. These findings suggest differences in the time course of automatic detection of a change in voice identity. In addition, they suggest that stimulus complexity modulates the magnitude of the orienting response to SGV and NSV stimuli, extending previous findings on self-voice processing.This work was supported by Grant Numbers IF/00334/2012, PTDC/PSI-PCL/116626/2010, and PTDC/MHN-PCN/3606/2012, funded by the Fundacao para a Ciencia e a Tecnologia (FCT, Portugal) and the Fundo Europeu de Desenvolvimento Regional through the European programs Quadro de Referencia Estrategico Nacional and Programa Operacional Factores de Competitividade, awarded to A.P.P., and by FCT Doctoral Grant Number SFRH/BD/77681/2011, awarded to T.C.info:eu-repo/semantics/publishedVersio

Investigating the global genomic diversity of Escherichia coli using a multi-genome DNA microarray platform with novel gene prediction strategies

<p>Abstract</p> <p>Background</p> <p>The gene content of a diverse group of 183 unique <it>Escherichia coli </it>and <it>Shigella </it>isolates was determined using the Affymetrix GeneChip^®<it>E. coli </it>Genome 2.0 Array, originally designed for transcriptome analysis, as a genotyping tool. The probe set design utilized by this array provided the opportunity to determine the gene content of each strain very accurately and reliably. This array constitutes 10,112 independent genes representing four individual <it>E. coli </it>genomes, therefore providing the ability to survey genes of several different pathogen types. The entire ECOR collection, 80 EHEC-like isolates, and a diverse set of isolates from our FDA strain repository were included in our analysis.</p> <p>Results</p> <p>From this study we were able to define sets of genes that correspond to, and therefore define, the EHEC pathogen type. Furthermore, our sampling of 63 unique strains of O157:H7 showed the ability of this array to discriminate between closely related strains. We found that individual strains of O157:H7 differed, on average, by 197 probe sets. Finally, we describe an analysis method that utilizes the power of the probe sets to determine accurately the presence/absence of each gene represented on this array.</p> <p>Conclusions</p> <p>These elements provide insights into understanding the microbial diversity that exists within extant <it>E. coli </it>populations. Moreover, these data demonstrate that this novel microarray-based analysis is a powerful tool in the field of molecular epidemiology and the newly emerging field of microbial forensics.</p

Array CGH Phylogeny: How accurate are Comparative Genomic Hybridization-based trees?

<p>Abstract</p> <p>Background</p> <p>Array-based Comparative Genomic Hybridization (CGH) data have been used to infer phylogenetic relationships. However, the reliability of array CGH analysis to determine evolutionary relationships has not been well established. In most CGH work, all species and strains are compared to a single reference species, whose genome was used to design the array. In the accompanying work, we critically evaluated CGH-based phylogeny using simulated competitive hybridization data. This work showed that a limited number of conditions, principally the tree topology and placement of the reference taxon in the tree, had a strong effect on the ability to recover the correct tree topology. Here, we add to our simulation study by testing the use of CGH as a phylogenetic tool with experimental CGH data from competitive hybridizations between <it>N. crassa </it>and other <it>Neurospora </it>species. In the discussion, we add to our empirical study of <it>Neurospora </it>by reanalyzing of data from a previous CGH phylogenetic analysis of the yeast <it>sensu stricto </it>complex.</p> <p>Results</p> <p>Array ratio data for <it>Neurospora </it>and related species were normalized with loess, robust spline, and linear ratio based methods, and then used to construct Neighbor-Joining and parsimony trees. These trees were compared to published phylogenetic analyses for <it>Neurospora </it>based on multilocus sequence analysis (MLSA). For the <it>Neurospora </it>dataset, the best combination of methods resulted in recovery of the MLSA tree topology less than half the time. Our reanalysis of a yeast dataset found that trees identical to established phylogeny were recovered only by pruning taxa - including the reference taxon - from the analysis.</p> <p>Conclusion</p> <p>Our results indicate that CGH data can be problematic for phylogenetic analysis. Success fluctuates based on the methods utilized to construct the tree and the taxa included. Selective pruning of the taxa improves the results - an impractical approach for normal phylogenetic analysis. From the more successful methods we make suggestions on the normalization and post-normalization methods that work best in estimating genetic distance between taxa.</p

Specific patterns of gene space organisation revealed in wheat by using the combination of barley and wheat genomic resources

<p>Abstract</p> <p>Background</p> <p>Because of its size, allohexaploid nature and high repeat content, the wheat genome has always been perceived as too complex for efficient molecular studies. We recently constructed the first physical map of a wheat chromosome (3B). However gene mapping is still laborious in wheat because of high redundancy between the three homoeologous genomes. In contrast, in the closely related diploid species, barley, numerous gene-based markers have been developed. This study aims at combining the unique genomic resources developed in wheat and barley to decipher the organisation of gene space on wheat chromosome 3B.</p> <p>Results</p> <p>Three dimensional pools of the minimal tiling path of wheat chromosome 3B physical map were hybridised to a barley Agilent 15K expression microarray. This led to the fine mapping of 738 barley orthologous genes on wheat chromosome 3B. In addition, comparative analyses revealed that 68% of the genes identified were syntenic between the wheat chromosome 3B and barley chromosome 3 H and 59% between wheat chromosome 3B and rice chromosome 1, together with some wheat-specific rearrangements. Finally, it indicated an increasing gradient of gene density from the centromere to the telomeres positively correlated with the number of genes clustered in islands on wheat chromosome 3B.</p> <p>Conclusion</p> <p>Our study shows that novel structural genomics resources now available in wheat and barley can be combined efficiently to overcome specific problems of genetic anchoring of physical contigs in wheat and to perform high-resolution comparative analyses with rice for deciphering the organisation of the wheat gene space.</p

Caracterização do gene vip3A e toxicidade da proteína Vip3Aa50 à lagarta-do-cartucho e à lagarta-da-soja

O objetivo deste trabalho foi caracterizar o gene vip3A de Bacillus thuringiensis e verificar a toxicidade da proteína Vip3Aa50 a larvas da lagarta-do-cartucho (Spodoptera frugiperda) e da lagarta-da-soja (Anticarsia gemmatalis). O gene vip3A foi amplificado por PCR, com iniciadores específicos, e gerou um fragmento de 2.370 pb. Esse fragmento foi clonado em vetor pGEM-T Easy e, em seguida, sequenciado, subclonado em vetor de expressão pET-28a (+) e inserido em células de Escherichia coli BL21 (DE3). A expressão da proteína Vip3Aa50 foi induzida por isopropil-β-D-1-tiogalactopiranosídeo (IPTG), visualizada em SDS-PAGE e detectada por "Western blot". Os ensaios de toxicidade revelaram alta atividade da proteína Vip3Aa50 contra as larvas neonatas da lagarta-da-soja e da lagarta-do-cartucho, com CL50 de 20,3 e 79,6 ng cm-2, respectivamente. O gene vip3Aa50 é um novo gene da classe vip3A